5-Fluorouridine (5-FUrd, CAS 316-46-1, C₉H₁₁FN₂O₆, MW 262.19 g/mol) is a fluorinated nucleoside analog — the riboside form of 5-fluorouracil (5-FU), one of the most important cancer chemotherapy agents on the WHO Essential Medicines List. The defining structural feature is the substitution of a fluorine atom for hydrogen at the C5 position of the uracil base. Fluorine acts as a hydrogen bioisostere because its van der Waals radius (1.47 Å) is only marginally larger than hydrogen (1.20 Å), and the C–F bond length (1.35 Å) closely approximates the C–H bond length (1.09 Å). This near-identical steric profile means that enzymes — particularly thymidylate synthase (TS) and uracil-metabolizing enzymes — cannot sterically distinguish 5-fluorouridine from natural uridine at their active sites. The molecule enters the pyrimidine nucleotide metabolic pathway as a molecular Trojan horse: 5-FUrd is converted by uridine-cytidine kinase to 5-FUMP, then to 5-FUTP (incorporated into RNA, disrupting RNA processing and function) and 5-FdUMP (a potent inhibitor of TS). The critical difference is that while hydrogen can be abstracted from C5 of uracil during the TS catalytic cycle, the C–F bond is essentially unbreakable under physiological conditions (bond dissociation energy ~130 kcal/mol vs C–H ~105 kcal/mol). This means the fluorine atom is recognized as hydrogen — enabling enzyme binding — but the C–F bond cannot be cleaved, trapping the enzyme-substrate complex in a covalent ternary dead-end complex. This "deception" mechanism — F mimics H but C–F is unbreakable — is the foundation of fluorinated drug design and is formally known as mechanism-based enzyme inhibition or suicide inhibition. UPOR Biotech supplies synthetic 5-fluorouridine with full ¹H-NMR confirmation of C5-fluorine substitution (characteristic C6-H doublet, J_H-F ~6.5 Hz) and ≥99% HPLC purity per lot.

5-Fluorouridine (5-FUrd) is the direct riboside metabolite of 5-fluorouracil (5-FU) and is central to 5-FU’s antineoplastic mechanism of action. The metabolic activation pathway proceeds as follows: 5-FU is first converted to 5-FUrd by uridine phosphorylase (UPase, EC 2.4.2.3), which catalyzes the reversible phosphorolysis of 5-FU with ribose-1-phosphate. 5-FUrd is then phosphorylated by uridine-cytidine kinase (UCK, EC 2.7.1.48) to 5-fluorouridine monophosphate (5-FUMP). From 5-FUMP, the pathway bifurcates: (1) 5-FUMP is sequentially phosphorylated to 5-FUDP and then 5-fluorouridine triphosphate (5-FUTP), which is recognized by RNA polymerases as a uridine analog and incorporated into all classes of RNA (mRNA, rRNA, tRNA, snRNA), causing widespread disruption of RNA processing, splicing, polyadenylation, and translation — this RNA-mediated cytotoxicity is now recognized as the dominant mechanism of 5-FU antitumor activity; (2) 5-FUMP can also be converted via ribonucleotide reductase (RNR) and thymidylate synthase pathway intermediates to 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), which forms a stable covalent ternary complex with thymidylate synthase (TS) and 5,10-methylenetetrahydrofolate (CH₂-THF), irreversibly inhibiting TS — the sole de novo source of dTMP (thymidylate) in cells — leading to thymineless death via DNA synthesis arrest and dUTP misincorporation. Administering 5-FUrd directly as the pre-formed riboside bypasses the uridine phosphorylase step, providing a more direct route to the active 5-FUMP intermediate and potentially overcoming UPase-mediated resistance mechanisms. This makes 5-FUrd an invaluable research tool for studying 5-FU metabolism, resistance pathways, and RNA-directed fluoropyrimidine pharmacology independent of the rate-limiting UPase activation step. 5-FU itself is on the WHO Model List of Essential Medicines and remains a first-line component of chemotherapy regimens for colorectal, breast, gastric, head and neck, and pancreatic cancers worldwide.

The thymidylate synthase (TS) suicide inhibition by 5-FUrd-derived 5-FdUMP is the textbook example of mechanism-based enzyme inactivation — a multi-step catalytic trap that converts the enzyme’s normal catalytic machinery against itself. The normal TS catalytic cycle: TS catalyzes the reductive methylation of dUMP to dTMP using CH₂-THF as both the methylene donor and the hydride source. The catalytic cysteine (Cys195 in human TS) performs a nucleophilic attack on C6 of dUMP, forming a covalent enzyme-substrate enolate intermediate. The enolate at C5 then attacks the methylene of CH₂-THF, followed by hydride transfer from THF and β-elimination to release dTMP and regenerate the free enzyme. The suicide inhibition sequence with 5-FdUMP: (1) The catalytic cysteine attacks C6 of 5-FdUMP exactly as it would with natural dUMP. (2) The C5 enolate attacks CH₂-THF as normal. (3) BUT — when the enzyme attempts to abstract the C5 proton (hydrogen) to complete β-elimination and product release, the fluorine atom (rather than hydrogen) occupies the C5 position. The C–F bond cannot be broken under the basic conditions of the enzyme active site (bond dissociation energy ~130 kcal/mol vs C–H ~105 kcal/mol). (4) The catalytic cycle stalls irreversibly at the covalent ternary complex: TS–5-FdUMP–CH₂-THF. This adduct is kinetically inert — the enzyme is permanently inactivated (k_inact ~0.1 min¹, K_i ~1 nM). Unlike competitive inhibitors that dissociate, suicide inhibition permanently removes the enzyme from the catalytic pool; recovery of TS activity requires de novo protein synthesis. This is why TS is a rate-limiting target — TS is the sole cellular source of dTMP; its irreversible inactivation triggers dTTP pool depletion, dUTP accumulation (from dUTPase saturation), and catastrophic uracil misincorporation into DNA during replication — a process termed "thymineless death." The exquisite specificity of this mechanism depends entirely on fluorine’s bioisosteric mimicry of hydrogen: the enzyme recognizes 5-FdUMP as its natural substrate dUMP, initiates catalysis, and is then trapped by the unbreakable C–F bond. This principle — fluorine as a catalytic poison disguised as hydrogen — extends far beyond TS to inform the design of fluorinated mechanism-based inhibitors across medicinal chemistry.

5-Fluorouridine (5-FUrd), 5-fluoro-2'-deoxyuridine (5-FdUrd, floxuridine), and 5-fluorouracil (5-FU) represent three distinct nodes in the fluoropyrimidine pharmacological network, each with different metabolic entry points and therapeutic profiles: (1) 5-Fluorouracil (5-FU, parent compound) — requires multi-step anabolic activation: 5-FU → 5-FUrd (via uridine phosphorylase) → 5-FUMP → 5-FUDP → 5-FUTP (RNA incorporation) and 5-FdUMP (TS inhibition). The UPase step is rate-limiting and subject to resistance via UPase downregulation. Clinical administration: intravenous infusion for colorectal, breast, head/neck, and gastric cancers. (2) 5-Fluorouridine (5-FUrd, the riboside) — bypasses the UPase step; directly phosphorylated by uridine-cytidine kinase to 5-FUMP. Preferentially channels into the RNA-directed cytotoxicity pathway (5-FUTP incorporation). Primary use: research tool for studying RNA-mediated 5-FU toxicity, UPase-independent activation, and antisense oligonucleotide modification. (3) 5-Fluoro-2'-deoxyuridine (5-FdUrd, floxuridine, FUDR) — the 2'-deoxyriboside; directly phosphorylated by thymidine kinase to 5-FdUMP (the TS inhibitor) without requiring ribonucleotide reductase-mediated conversion. Preferentially channels into DNA-directed cytotoxicity (TS inhibition → thymineless death). Clinical use: hepatic arterial infusion for colorectal liver metastases (high first-pass hepatic extraction, >90%). (4) Capecitabine — oral prodrug: 5'-deoxy-5-fluorocytidine → 5'-deoxy-5-fluorouridine (via carboxylesterase/cytidine deaminase in liver) → 5-FU (via thymidine phosphorylase, preferentially in tumor tissue). Designed for tumor-selective activation. Key research distinction: 5-FUrd is the riboside, targeting RNA metabolism and serving as the metabolic bridge between 5-FU and 5-FUMP. It is the preferred reagent for investigating the RNA-directed pharmacology of fluoropyrimidines, independent of the competing DNA-directed 5-FdUrd/5-FdUMP pathway. Researchers studying the relative contributions of RNA damage vs TS inhibition to 5-FU cytotoxicity use 5-FUrd and 5-FdUrd as orthogonal probes to dissect the two pathways.

Every shipment of 5-fluorouridine from UPOR Biotech includes: COA (HPLC purity ≥99.0% or ≥98.0%, full impurity profile, specific optical rotation, heavy metals ≤10 ppm, residual solvents per USP <467> / ICH Q3C, microbial panel per USP <61>/<62>, endotoxins ≤0.5 EU/mg per USP <85>), MSDS/SDS (Safety Data Sheet compliant with GHS/CLP regulations), HPLC Chromatogram (signed and dated by QC analyst), ¹H-NMR Spectrum (with full peak assignments confirming C5-fluoro substitution via characteristic C6-H doublet, J_H-F ~6.5 Hz), IR Spectrum, Elemental Analysis Report (C, H, N, F), BSE/TSE-Free Statement, Residual Solvent Analysis (per USP <467> / ICH Q3C), Heavy Metals Analysis (USP <231>/<232>), Stability Data (real-time 2–8 °C and accelerated 25 °C/60%RH), and Complete Lot Traceability from synthesis batch to finished product. Additional documentation available upon request: custom QC testing, method validation packages, technical data sheets (TDS), and impurity reference standards. Free sample available for qualified B2B buyers (research institutions, pharmaceutical companies, CROs). MOQ: 1 g for research evaluation; bulk from 100 g to multi-kilogram scale. ISO 9001:2015 and c-GMP compliant manufacturing. All documentation provided in English.